JENS LÜDERS
Microtubule organisation
O
ur overall goal is to reach a comprehensive understanding of how cells generate, maintain,
and remodel the microtubule cytoskeleton during cell cycle and cell differentiation. Defects
in the structure and function of the microtubule cytoskeleton are linked to cancer and certain
developmental disorders. To ensure proper microtubule organisation, cells control where and when
microtubules are made. Microtubule organising centres, such as the animal centrosome, participate
in this regulation by providing sites that nucleate the polymerisation of new microtubules. These
sites are formed by interaction with a large, multi-subunit protein complex, the g-tubulin ring
complex, which functions as microtubule nucleator. Microtubule nucleation sites provide a unique
molecular environment, not only for the control of microtubule nucleation but also for regulating
microtubule behaviour, and thus are central to our understanding of microtubule organisation.
However, the exact molecular composition of microtubule nucleation sites and the spatio-temporal
regulation of their assembly are poorly understood. To address this issue, we study centrosomal and
non-centrosomal microtubule nucleation pathways in several cell types by identifying the molecular
players and by characterising their function and regulation in vitro and in vivo.
In proliferating cells, the major microtubule organising centre
(MTOC), the centrosome, organises the mitotic spindle. Numerical and functional centrosome abnormalities including aberrant
size and shape, and microtubule (MT) nucleation activity are
frequently found in cancer cells. Such centrosomal defects can
impair proper spindle assembly and function and result in genomic instability. In addition to centrosomal MT nucleation, our
previous work and studies by others show that proper mitotic
spindle assembly requires MT nucleation from non-centrosomal
sites. Non-centrosomal nucleation pathways are poorly characterised, but might be crucial for rapidly dividing cancer cells.
These pathways may therefore provide great potential for future anti-cancer therapies.
Centrosomal microtubule nucleation in mitosis
Figure 1. Plk1 activity is required for localisation of the
γ-tubulin targeting factor GCP-WD to centrosomes and spindle
microtubules. HeLa cells treated with monastrol or the Plk1
inhibitor BI2536 were fixed and immunostained for GCP-WD and
α-tubulin to label microtubules. DAPI was used to label DNA. Both
treatments produce monopolar spindles. Monastrol treatment,
however, does not affect centrosome accumulation of GCP-WD,
whereas BI2536 almost completely blocks recruitment of GCP-WD
to the centrosomes.
The centrosome comprises a pair of barrel-shaped centrioles
surrounded by a dense proteinaceous matrix, the pericentriolar material (PCM). The nucleation of MT polymerisation occurs within the PCM and requires the recruitment of γ-tubulin
ring complexes (γTuRCs) from the cytoplasm. These complexes
contain γ-tubulin, a paralogue of α- and β-tubulin that is not
incorporated into the MT polymer but functions as MT nucleator. We have previously shown that the interaction of γTuRCs
with centrosomes is mediated by a protein named GCP-WD (also
22 2008 Scientific Report Cell and Developmental Biology Programme
RESEARCH GROUP MEMBERS
Principal Investigator Jens Lüders Postdoctoral Fellow Marco Archinti Research Assistant
Cristina Lacasa Visiting Students Florian Baier (Germany), Sabine Klischies (Germany), Leila
Njimoluh (USA)
known as NEDD1) (Lüders et al, 2006; Haren et al, 2006). When
cells prepare for mitosis in late G2 phase of the cell cycle, size
and microtubule nucleating activity of the duplicated centrosomes increase. This is accomplished by the recruitment of additional PCM to the centrosomes, including proteins involved in
microtubule nucleation and organisation, such as γ-tubulin. This
process, also termed centrosome maturation, is critical for the
function of centrosomes as MTOCs in mitosis, and depends on
the activity of mitotic kinases such as Polo-like kinase 1 (Plk1).
Interference with Plk1 function by RNAi or specific inhibitors
prevents the recruitment of γ-tubulin to mitotic centrosomes,
thereby essentially inactivating their MT nucleation activity,
and impairing bipolar spindle formation. Plk1 inhibitors are currently being studied in clinical trials as potential agents for cancer therapy. To date, a Plk1 substrate that controls γ-tubulin
recruitment in a phosphorylation-dependent manner has not
been identified.
We discovered that Plk1 associates with GCP-WD, the γ-tubulin
targeting factor, and Plk1 activity contributes to mitotic phosphorylation of GCP-WD (Haren et al, 2009, in preparation). Plk1
depletion or inhibition revealed that accumulation of γ-tubulin
at centrosomes is regulated by controlling the levels of centrosomal GCP-WD (Figure 1). Surprisingly, GCP-WD mutants that are
defective in Plk1 binding and phosphorylation still accumulate
at mitotic centrosomes and recruit γ-tubulin. At present, we are
studying whether the Plk1-dependent phosphorylation of GCPWD serves other functions and whether it affects spindle assembly and function.
Interestingly, our studies revealed that Plk1 also controls the
recruitment of other PCM proteins implicated in centrosomal
γ-tubulin attachment. Our results support a model in which
Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes
is regulated upstream of GCP-WD, and involves multiple PCM
proteins and potentially multiple Plk1 substrates (Haren et al,
2009, in preparation). Our next goal is to identify these substrates and, through phospho-mutant analysis, shed light on the
Plk1-dependent pathway that triggers centrosome maturation
and γTuRC recruitment.
To study the role of Plk1 and other mitotic kinases in the regulation of microtubule nucleation during spindle formation, we
have initiated a collaboration project with Carme Caelles and
Joan Roig, researchers in the Molecular Medicine Programme at
IRB Barcelona.
In addition to the general importance of this pathway for proliferating cells, two of the proteins involved in γ-tubulin recruitment to centrosomes have recently been implicated in micro-
Cell and Developmental Biology Programme 2008 Scientific Report 23
cephaly, a condition associated with certain neurodevelopmental disorders. Mutations in the genes of Cep215/Cdk5Rap2 and
pericentrin cause primary microcephaly and Seckel syndrome,
respectively. In both cases, affected individuals have abnormally
small brains and show mental retardation. It has been speculated that microcephaly is caused by defects in the proliferation of neuronal progenitor cells, but mechanistic insight is still
missing. We hope that our studies will contribute to a better
understanding of these diseases.
Non-centrosomal microtubule nucleation in mitosis
Figure 2. Mitotic phosphorylation of GCP-WD is required
for spindle targeting but not for centrosome targeting.
HeLa cells were cotransfected with a plasmid expressing
shRNA targeting endogenous GCP-WD and plasmids
expressing either RNAi-insensitive GCP-WD-MycHis or the
phosphorylation mutant GCP-WD S418A-MycHis. Cells were
stained with anti-Myc and anti-α-tubulin antibodies and DAPI
to visualise DNA.
Figure 3. ‘Amplification model’ for the function of the
γTuRC in spindle assembly. Microtubules are nucleated
by γTuRC at the two centrosomes (1) and near the mitotic
chromatin (2). A third pathway depends on γTuRC bound to
the sides of existing spindle microtubules (3). This pathway
‘amplifies’ the process of spindle assembly by nucleating
additional microtubules where needed, within the forming
spindle. Correct orientation is achieved by directional
nucleation, or by motor-mediated orientation soon after
nucleation. Minus end-bound γTuRC facilitates the capture
of free microtubules by directing their ends to the sides
of other microtubules (4). γTuRCs are depicted in green,
microtubules in red, and chromosomes in blue.
γTuRCs typically interact with MT minus ends at centrosomes,
where they nucleate and stabilise MTs. However, a large
number of γTuRCs are also found in the soluble fraction of the
cytoplasm and associated with mitotic spindle microtubules.
In mitosis, these γTuRCs participate in non-centrosomal MT nucleation pathways, such as the chromatin-mediated nucleation
pathway, which is controlled by the small GTPase Ran. Recently, we discovered that mitotic phosphorylation of GCP-WD at
a Cdk1 consensus phosphorylation site targets the protein to a
subset of spindle MTs (Figure 2). Mutation of this phosphorylation site interferes specifically with the recruitment of γTuRC to
spindle MTs.
The lack of spindle-associated γTuRCs results in defective spindles that assemble less efficiently and have a lower density of
MTs, but does not seem to affect centrosomal or chromatinmediated nucleation (Lüders et al, 2006). On the basis of these
findings, we proposed that spindle-bound γTuRC is required
to nucleate additional MTs within the spindle to allow proper
spindle formation. Studies in Drosophila have identified components of a protein complex termed augmin, which function
upstream of GCP-WD in mediating the association of γTuRCs with
mitotic spindle microtubules (Goshima et al, 2008). Together,
these studies support a model in which γTuRCs are recruited to
the sides of pre-existing spindle MTs to nucleate additional MTs,
thereby “amplifying” MT nucleation and promoting spindle formation (Lüders and Stearns, 2007; Figure 3).
We have started to characterise this new pathway in human
cells. One of our goals is to achieve a molecular understanding
of how these novel, non-centrosomal MT nucleation sites are assembled. Using a cell line that stably expresses γ-tubulin fused
to photoactivatable GFP, we demonstrated that, compared to
the interaction of γ-TuRCs with centrosomes, the interaction of
γTuRCs with spindle microtubules is much more dynamic (Archinti, Lacasa and Lüders, in preparation), thereby hindering direct
analysis in living cells. We are using bioinformatics, yeast twohybrid screening, biochemical and RNAi-based approaches to
identify the components of this pathway. We have already identified several candidate genes, which we are currently analysing
for a role in non-centrosomal MT nucleation within the spindle.
In addition, we have generated tools, such as stable cell lines
and antibodies, for the analysis of this pathway in vitro and in
vivo.
Microtubule nucleation during differentiation
During the differentiation of muscle cells, myoblasts fuse to
form multi-nucleated myotubes. This process involves a reor-
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ganisation of the MT network from a radial, centrosome-based
MT array to an elongated array composed of parallel MTs in myotubes. Interestingly, centrosomes degenerate during myotube
formation and differentiation. Several PCM proteins, including
γ-tubulin, redistribute to the surface of the nuclear envelope,
which functions as MTOC. Whether the nuclear envelope-associated MTOC is critical for myotube formation or differentiation
is not known.
Using lentivirus-mediated infection and expression of shRNA to
deplete endogenous GCP-WD in mouse C2C12 cells, an in vitro
model for muscle cell differentiation, we found that, in contrast
to its role in centrosome attachment, the γ-tubulin targeting factor GCP-WD is not required for nuclear envelope localisation of
γTuRC. Moreover, a few days after differentiation is initiated,
GCP-WD expression is down-regulated, thereby suggesting that its
function is not required in differentiated muscle cells. γ-tubulin
expression remains relatively constant, suggesting that the γTuRC
participates in MT organisation throughout the differentiation
process. To address this question, we have also established conditions for efficient RNAi-mediated depletion of γ-tubulin in muscle
cells. These studies will provide the first insight into the role of
non-centrosomal MT nucleation pathways in the reorganisation of
the MT cytoskeleton during cellular differentiation.
SCIENTIFIC OUTPUT
Research networks and grants
Microtubule organizing centers and microtubule nucleation in
mitosis (MTOC function)
European Commission, PEOPLE-2007-4-3-IRG (2008-2012)
Principal investigator: Jens Lüders
Regulation of microtubule nucleation through phosphorylation
Carme Caelles and Joan Roig, Molecular Medicine Programme, IRB
Barcelona (Barcelona, Spain)
Collaborations
Recruitment of γ-tubulin complexes to mitotic centrosomes
Andreas Merdes and Laurence Haren, Institut de Sciences et
Technologies du Médicament de Toulouse, Centre National de la
Recherche Scientifique/Pierre Fabre (Toulouse, France)
Cell and Developmental Biology Programme 2008 Scientific Report 25